Use of radioisotopes for rapid identification of microorganisms

ABSTRACT

There is disclosed a process for the rapid identification of a microorganism and an apparatus for performing the method. The process comprises inoculating a number of different  14  C labeled substrates with an unknown organism, at least some of said  14  C labeled substrates being capable of being metabolized to  14  CO 2  by certain specific microorganisms. The substrates are incubated for a time sufficient to cause metabolic breakdown of at least some of said substrates resulting in the production of  14  CO 2  and analysis for radioactivity in the  14  CO 2  which is evolved from those substrates which are metabolized is performed to determine which substrates are metabolized and which substrates are not metabolized by the unknown microorganisms. There is thereby obtained a substrate radiorespirometric profile for the unknown microorganisms which is compared to standard radiorespirometric profiles for known organisms to determine to which standard radiorespirometric profile the radiorespirometric profile for said unknown microorganisms corresponds. 
     The apparatus includes trays for incubating the organisms in a selection of substrates, the trays having covers for collecting the evolved  14  CO 2 , conveyor means for transporting the covers to particle detectors, drive means for stepwise advancing the covers to sequentially expose the collection zones thereon to the detectors, counters for accumulating records of radiation levels, a data file of organism and substrate profiles, and means for comparing the profiles with the accumulated counts.

This invention relates to the use of radioisotopes for the rapididentification of microorganisms. More particularly, this inventionrelates to the identification of an unknown organism by obtaining aradiorespirometric profile of the unknown organism and comparing thisradiorespirometric profile to standard radiorespirometric profilesobtained on known organisms to determine to which one it willcorrespond. By "radiorespirometric" is meant the measurement ofmetabolism of a microorganism which results in the evolution ofradioactive gas from a radioactive substrate.

The term "microorganism" as used throughout this application refers toOrders I to X bacteria, mycoplasma, actinomycetes and fungi which arelisted in Bergey's Manual of Determinative Bacteriology, SeventhEdition, Williams & Wilkins Co., 1957, by Breed, Robert S., E.G.D.Murray and Nathan, R. Smith, the disclosure of which is incorporatedherein by reference.

The conventional identification of these microorganisms is atime-consuming and laborious process. Thus, for example, to identify anorganism which is present in urine, stools, blood, spinal fluid, etc.,the specimen is first streaked on agar plates or processed by otherknown techniques to obtain isolated colonies of the microorganism. Afterthe unknown organism is isolated, it must then be subjected to a batteryof tests to identify it. These tests include colony and cell morphology,stain characteristics, susceptibility to antimetabolites and serologicaland biochemical properties. Since many of the tests require relativelylong incubation times, it frequently requires a minimum of 18 hoursafter isolation to arrive at a positive identification of the unknownmicroorganism. A method of microbial identification which would minimizethe time required to make the identification would be an obviousadvantage in the treatment of a patient since this would permit theattending physician to, e.g., prescribe the correct type and dosage ofan antibiotic.

Rapid means for the identification of microorganisms would also bevaluable in other areas such as to identify microorganisms present infood, drugs, spices, cosmetics, wine, beer, potable water, waste water,air, soil, etc.

Accordingly, it is a principal object of this invention to provide ameans for the rapid identification of microorganisms.

According to the present invention, there is provided a process for therapid identification of a microorganism which comprises inoculating anumber of different ¹⁴ C specifically or uniformly labeled substrates,i.e., a substrate which contains at least some carbon atoms having anatomic weight of 14, with an unknown organism. Each of the ¹⁴ C labeledsubstrates is capable of being metabolized to ¹⁴ CO₂ by certain specificmicroorganisms. All of the substrates are then incubated for apredetermined length of time sufficient to cause breakdown of at leastsome of the substrates resulting in the production of ¹⁴ CO₂.

Metabolism is defined as fermentative or oxidative metabolism via knownand/or unknown pathways, which affects a ¹⁴ C atom of the substrate toproduce ¹⁴ CO₂. The extent of metabolism of substrates by themicroorganism is determined by analyzing radioactivity which is evolved.If no ¹⁴ CO₂ is evolved as indicated by the analysis for radioactivity,then it is known that those substrates are not metabolized at the ¹⁴ Catom to produce ¹⁴ CO₂ by the particular unknown microorganism withinthe given incubation time. By determining the quantity of ¹⁴ CO₂produced from each ¹⁴ C labeled substrate, within a given time intervalby the unknown microorganism, there is obtained a substrateradiorespirometric profile which serves as a fingerprint of the unknownmicroorganism. This radiorespirometric profile is then compared tostandard radiorespirometric profiles which have been obtained in thesame manner on known microorganisms. By determining which standardradiorespirometric profile the radiorespirometric profile of the unknownmicroorganism corresponds to, one is able to identify the unknownorganism.

The process described hereinabove for the rapid identification ofmicroorganisms by the use of radioisotopes may be conducted in anapparatus which comprises a means for containing a number of differentand separate ¹⁴ C labeled substrates, means for inoculating each of the¹⁴ C labeled substrates with an unknown microorganism, means forcollecting radioactive gas which is produced as a result of metabolismof the ¹⁴ C labeled substrates and means for analyzing any gas obtainedfor radioactivity to determine whether metabolism occurred in eachspecific ¹⁴ C labeled medium.

The invention will be more fully described by reference to theaccompanying drawings wherein:

FIG. 1 is a flow diagram showing the process of this invention;

FIG. 2 is a perspective view of a microculture tray, partly in section,which may be used in the practice of this invention;

FIG. 3 is a sectional view taken along line 3--3 of FIG. 2;

FIG. 4 is a view similar to FIG. 3 showing the tray in combination witha cover;

FIGS. 5, 6 and 7 are graphs showing the rate and quantity of radioactiveCO₂ gas evolved by different microorganisms in the presence of various¹⁴ C labeled substrates;

FIGS. 8 and 9 illustrate the range of cumulative evolved radioactive CO₂gas obtained in a series of repetitions of two different microorganismson various substrates;

FIG. 10 is an exploded perspective view of an incubation unit assembly;

FIG. 11 is a side elevation, in section of the assembly of FIG. 10;

FIG. 12 is a perspective view of a plurality of the assemblies of FIGS.10 and 11 stacked for incubation;

FIGS. 13 and 14 are perspective views of two forms of collectionmaterial carriers;

FIG. 15 is a perspective view, partially schematic, of a counting andanalysis system usable with the assemblies of FIGS. 10-14;

FIG. 16 is a schematic diagram of an analysis and control system usablewith the apparatus of FIGS. 10-15;

FIG. 17 is a side elevation of a portion of the tray of FIGS. 13 or 14;

FIG. 18 is a sectional view of a portion of the tray and guide rail ofthe apparatus of FIG. 15; and

FIG. 19 is a partial plan view, in partial section of the apparatus ofFIG. 18.

In accordance with the practice of this invention, standardradiorespirometric profiles are prepared for known microorganisms.Cultures of known microorganisms may be obtained from repositories suchas the Center for Disease Control in Atlanta, Georgia (CDC) and AmericanType Culture Collection. Set forth in Bergey's Manual of DeterminativeBacteriology, Orders I to X, are a number of microorganisms for whichstandard radiorespirometric profiles may be prepared. In some cases itmay be sufficient to merely identify the microorganism by genus.However, in other cases, it may be desirable to identify the particularspecies of microorganism within the genus. Therefore, in accordance withthis invention, it is sufficient that the radiorespirometric profileidentify the microorganism as one which falls within a particular groupwhich is differentiated from microorganisms falling within other groups.Species for which experimental data have been obtained include thosemicroorganisms listed in Table 1.

TABLE 1

escherichia coli

Alkalscens-Dispar

Shigella dysenteriae

Shigella, flexneri

Shigelli sonnei

Edwardsiella

Salmonella Gp B

salmonella paratyphi A

salmonella choleraesuis

Salmonella typhi

Salmonella pullorum

Salmonella galinarum

Arizona (lactose -)

Arizona (lactose +)

Citrobacter

Klebsiella pneumonia

Klebsiella ozaenae

Klebsiella rhinoschleromatis

Enterobacter aerogenes

Enterobacter hafnia

Enterobacter cloacae

Enterobacter liquefaciens

Serratia marcescens

Proteus vulgaris

Proteus mirabilis

Proteus morganii

Proteus rettgeri

Providencia alcalifacens

Providencia stuartii

Pseudomonas aeruginosa

Pectobacterium carotovarum

Haemophilus influensae

Streptococcus faecalis

Each known organism is tested against a sufficient number of ¹⁴ Clabeled substrates to differentiate it from other microorganisms. Thesubstrate may contain one or more carbon atoms having an atomic weightof 14. Set forth in Table 2 are a number of ¹⁴ C labeled substrateswhich are commercially available and which have been tested. Each of thesubstrates set forth in Table 2 is assigned a numerical designation ornumbering code which is used in FIG. 7. However, the identificationmethod is not meant to be limited to only this list of substrates nor isthe position of ¹⁴ C carbon atom specified, the only carbon atom whichmay be labeled and used for the identification method. In this table,the designation "UL" indicates that the substrate is uniformly labeled,i.e., the ¹⁴ C label is evenly distributed to all carbon atom positionson the molecule. The numerical designation(s) preceding the names ofsome substrates indicates the position of the ¹⁴ C atom(s). Most of thesubstrates listed in Table 2 are in the form of the sodium salt,although the free acid form or other forms may also be used.

                  Table 2                                                         ______________________________________                                        Numbering                                                                     Code     .sup.14 C Labeled Substrates                                         ______________________________________                                         7       UL .sup.14 C urea                                                    10       1 .sup.14 C lactose                                                  18       15 .sup.14 C citrate                                                 19       UL .sup.14 C ornithine                                               20       UL .sup.14 C maltose                                                 21       UL .sup.14 C sucrose                                                 22       UL .sup.14 C D-glucose                                               23       1 .sup.14 C DL-lysine                                                24       1 .sup.14 C dulcitol                                                 25       UL .sup.14 C sorbitol                                                26       UL .sup.14 C D-xylose                                                27       UL .sup.14 C D-manitol                                               28       1 .sup.14 C ring DL-phenylalanine                                    29       UL .sup.14 C glycine                                                 30       UL .sup.14 C D-alanine                                               31       2 .sup.14 C xanthine                                                 32       UL L-alanine                                                         33       UL .sup.14 C formate                                                 34       UL .sup.14 C acetate                                                 35       UL .sup.14 C DL-lactate                                              38       1 .sup.14 C fumarate                                                 39       UL .sup.14 C malate                                                  40       1 .sup.14 C gluconate                                                41       UL .sup.14 C glutamate                                               42       UL .sup.14 C L-tyrosine                                              43       UL .sup.14 C L-threonine                                             44       UL .sup.14 C L-aspartic                                              47       1 .sup.14 C succinate                                                48       (ring 2 .sup.14 C) L-histidine                                       49       2 .sup.14 C (ring labeled) tryptophan                                50       1 .sup.14 C DL-leucine                                               51       1,3 .sup.14 C glycerol                                               52       1 .sup.14 C D-galactose                                              53       1 .sup.14 C D-mannose                                                54       Carbonyl .sup.14 C DL-methionine                                     55       1 .sup.14 C serine                                                   56       1,2 .sup.14 C oxalate                                                57       1 .sup.14 C DL-valine                                                58       1 .sup.14 C malonate                                                 59       4 .sup.14 C DL-aspartate                                             60       (guanido .sup.14 C) DL-arginine                                      61       UL .sup.14 C trehalose                                               62       2 .sup.14 C uracil                                                   63       UL .sup.14 C erythritol                                              64       1,4 .sup.14 C DL-tartrate                                            65       (carbonyl .sup.14 C) dextran                                         66       UL .sup.14 C starch                                                  67       UL .sup.14 C cellulose                                               73       1 .sup.14 C glucose                                                  74       6 .sup.14 C glucose                                                  75       1 .sup.14 C propionate                                               76       1 .sup.14 C butyric                                                  ______________________________________                                    

The production of ¹⁴ CO₂ from some substrates, such as lactose, isdependent upon the induction of enzymes by that substrate and,therefore, is time dependent. It is preferred to use substrates forwhich breakdown to produce ¹⁴ CO₂ is least time dependent--i.e.,substrates which do not require inducible enzymes for breakdown ortransport. The rapidness of the test is based upon constitutive enzymeswhich permit an organism to commence its action immediately.Conventional biochemical techniques utilize substrates which requiremany hours or days for metabolism. The radioisotopic procedure,therefore, does not conform to conventional results. Substrates whichgive positive fermentation results classically may be negative by theradiorespirometric method.

The media containing ¹⁴ C labeled substrates which are inoculated withthe microorganisms may be composed of a basal medium containing salts,buffers, growth factors, preservatives and a single ¹⁴ C labeledsubstrate or may comprise simply aqueous solutions of the substrates.These ¹⁴ C labeled media may conveniently be contained in a compact,partitioned or welled tray such as a microculture tray as shown in FIGS.2-5, or in a form separate from the microculture tray which is suitablefor addition to the microculture tray. The substrates may be present inthe wells of the tray in the form of liquid solutions, gels or in dryform--e.g., freeze dried. The substrate should be present in sufficientamount to provide a readily detectable amount of ¹⁴ CO₂ upon metabolism.Preferably, the substrate in each ¹⁴ C labeled medium will contain atleast four nanocuries of radioactivity.

A small amount of suspension of the microorganism is used to inoculateeach of the different ¹⁴ C labeled media to form a mixture of the ¹⁴ Clabeled medium and the organism. Inoculation may be performed byintroducing the suspension of microorganism manually or by automated ormass replicating techniques. If the ¹⁴ C labeled substrate is present indry form, the aqueous medium used to introduce the microorganism intothe means for containing the ¹⁴ C labeled substrate reconstitutes thesubstrate and forms a mixture of the ¹⁴ C labeled medium and theorganism.

Referring more specifically to FIGS. 2-5, there is shown a microculturetray 12 containing a plurality of wells 14. Each well 14 has a roundbottom 15. The wells may be dimensioned so as to have a diameter of, forexample, 1/2 inch and a height from top to bottom of 1/2 inch. Each wellcontains a medium 16 which includes a different ¹⁴ C labeled substrate.After the medium 16 in each well is inoculated with the suspension ofmicroorganism to form a mixture 17 of the medium and the microorganism,the wells are then covered with a means for collecting radioactive CO₂gas such as with a filter pad cover 18 containing a getter material,e.g., a solution of lithium hydroxide or barium hydroxide. The cover 18should create a tight seal of the well 14. A separate disc-shaped covercan be used to cover each well or, alternatively, a single sheet can beused to cover all of the wells in the tray 12. All of the inoculatedmedia are then incubated for a predetermined period of time, e.g., from10 minutes to four hours. During this incubation period, if the ¹⁴ Clabeled substrate contained in each medium is one which is metabolizedby the organism, ¹⁴ CO₂ will be evolved. This gas is collected by meansof the cover 18. Thus, if the cover 18 contains barium hydroxide, thegas which is evolved will react with the barium hydroxide to form Ba¹⁴CO₃ on the cover 18.

The covers 18 are then removed, dried and assayed for radioactivity. Theinstrument used for assaying for radioactivity can utilize severalGeiger tubes which will monitor and record the presence of carbon atomshaving an atomic weight of 14 contained on each of the covers 18--e.g.,a Gieger-Muller gas flow counter. The magnitude of radioactivity ismeasured for each of the getter covers and the results are recorded andused as a standard radiorespirometric profile for that organism. Aspecific system for accomplishing this is more fully discussedhereinafter.

A standard radiorespirometric profile may also be obtained by providinga radiorespirometric profile of an unknown organism in the mannerpreviously described for known organisms. The unknown organism may thenbe identified by means of conventional biochemical methods foridentification of unknown organisms. Thus, once the unknown organism hasbeen positively identified by conventional means, the radiorespirometricprofile for that organism may be used as a standard.

This invention may be used to identify microorganisms such as thosebelonging to the genera listed in Bergey's Manual of DeterminativeBacteriology, Orders I-X, including various groups of pathogenicbacteria such as enterics, fastidious gram negative organisms,anaerobes, neisseria, gram positive cocci, Mima herrilea Group, etc. Aradiorespirometric profile is obtained for the unknown organism in thesame manner as the standard radiorespirometric profiles are obtained. Apure culture is obtained from a sample of urine, blood, spinal fluid,food, water, air, etc. by streaking the sample material on an agar plateor by other known means. Incubation of these agar plates results in thegrowth of isolated colonies of organisms. Samples may then be taken fromthe individual colonies and suspended in an aqueous solution to preparethe inoculum for use in the practice of this invention.

The plates or other enrichment media used for obtaining the unknownorganism from an isolation specimen frequently contain inhibitors forthe growth of all microorganisms except certain species, so that onlythose species of microorganisms which are not inhibited will grow. Bythe use of such inhibitors of the isolation specimens or in the ¹⁴ Clabeled media, the isolation specimens may in certain cases be useddirectly for the inoculation of the ¹⁴ C labeled medium.

The size of the inoculum, i.e., the number of microorganism cellspresent in the mixture with the growth medium may vary over a relativelywide range without substantially influencing the results for a givensubstrate. It is, therefore, unnecessary to make precise andtime-consuming adjustments of inoculum size. In practice, the inoculumused in obtaining the data for FIGS. 5-9 was adjusted to an approximateoptical density of 1.0. Moreover, the concentration of the substrate maybe varied widely without substantially influencing the evolution of ¹⁴CO₂. Since large volumes of liquid retain significant quantities of CO₂,it is desirable to use a minimum amount of total liquid volume inperforming each test--e.g., from about 0.05-0.2 ml.

The magnitude of response as measured by the amount of ¹⁴ CO₂ evolvedduring the incubation reaction may be expressed in any suitable termssuch as counts per minute. The results for a particular microorganismassayed against each substrate may be recorded on a card using lines orholes to indicate the magnitude of radioactive response so that a visualcomparison may be made to a similar card prepared on an unknownorganism. Alternatively, the data may be stored in the memory bank of acomputer and the growth media profile obtained on an unknown organismmay be matched by the computer to determine whether it falls within theranges of response for the various standard radiorespirometric profiles.Statistical goodness of fit could be performed by the computer to givereliability of the identification. Further, they can be recorded simplyas positive or negative--e.g., below a certain number of counts perminute, the response will be considered negative, and above that numberof counts per minute the response will be considered positive. When theradiorespirometric profile for a given organism is expressed as eitherpositive or negative against a given substrate, it may be necessary toutilize a larger number of substrates than when the radiorespirometricprofile is expressed in actual numerical terms in order to differentiateit from radiorespirometric profiles for other microorganisms.

FIGS. 5 and 6 demonstrate how various species of microorganisms producea high level of ¹⁴ CO₂ from ¹⁴ C-glucose and ¹⁴ C-urea, respectively, asopposed to others which produce only low level evolution of ¹⁴ CO₂.Thus, in FIG. 5, all of the organisms set forth in Table 1 were testedunder identical conditions against the substrate ¹⁴ C-glucose. Thefailure of P. aeruginosa to produce substantial quantities of ¹⁴ CO₂from this substrate distinguishes it from the 29 other organisms tested.

Further, as shown in FIG. 6, testing of all of the organisms set forthin Table 1 against ¹⁴ C urea separated the genera of Proteus andKlebsiella from the remainder of the test group.

FIG. 7 shows the cumulative evolution of radioactivity from 21 differentsubstrates tested against E. coli. The identifying number to the rightof each curve refers to the identification of the substrate set forth inTable 3. Each curve was established by using three data points, one at15, 30 and 60 minutes.

The data obtained for each of FIGS. 5, 6, 7 and 8 was obtained by addingan inoculum comprising 0.1 ml. or one drop of suspension containing 10⁹cells of the various microorganisms per ml. of suspension. The inoculumwas added to 0.1 ml. or one drop of substrate in a microculture tray.The mixtures were then covered with getter covers and incubated for thetime indicated. The getter covers were then dried and assayed forradioactivity.

Incubation time does not appear to be a critical factor. As shown inFIG. 7, most substrates produced similar results after 15 minutes asobtained after 60 minutes. However, the timing of tests for unknown ispreferably about the same as used to establish standardradiorespirometric profiles.

FIGS. 8 and 9 illustrate the variations which may occur in repeatingassays for organisms against a number of different substrates. FIG. 8shows the ranges of values obtained for five repetitions of E. coliassayed against each substrate after one hour of incubation, and FIG. 9shows the ranges of values obtained for 12 repetitions of the bacteriumP. vulgaris assayed after one hour of incubation against the varioussubstrates.

Table 3 gives the radiorespirometric results obtained on 29 species ofmicroorganisms. In this table the substrate is identified in the lefthand column by the numbering code used in Table 2. The results set forthin Table 3 were obtained by inoculating the various substrates containedin wells of a microculture tray with one drop of an inoculum containingthe microorganism, the suspension having an optical density of about 1as measured on a spectrophotometer. The inoculum was added to one dropof each substrate and the mixture was covered with a getter covercontaining Ba(OH)₂ and incubated for 1 hour. The getter covers were thendried and assayed for radioactivity. Several repetitions for eachorganism were run against the various substrates. The highest and lowestvalues obtained for each organism on each substrate are set forth inTable 3. Thus, Table 3 sets forth radiorespirometric profiles for eachof these 29 species of microorganisms.

                                      TABLE 3                                     __________________________________________________________________________    Sub-  Escherichia                                                                             Alkalscens                                                                              Shigella                                            strate                                                                              coli      Dispar    dysenicriae                                         Code No.                                                                            1 hr (cpm)                                                                              1 hr. (cpm)                                                                             1 hr. (cpm)                                               Low  High Low  High Low  High                                           __________________________________________________________________________     7    <10  18        <10       <10                                            10    170  1,400                                                                              320  400  230  460                                            18    <10  12        <10       <10                                            19    460  4,700                                                                              2,200                                                                              5,000                                                                              <10  21                                             20    4,500                                                                              9,400                                                                              950  2,800                                                                              29   60                                             21    180  400  140  320  180  260                                            22    2,900                                                                              11,000                                                                             5,000                                                                              7,200                                                                              350  440                                            23    39   230  420  850       <10                                            24    <10  30   <10  64        <10                                            25    24   370  62   490  <10  26                                             26    <10  56   <10  280       <10                                            27    5,600                                                                              11,000                                                                             2,900                                                                              6,600                                                                              320  610                                            28    <10  12   <10  25        <10                                            29    6,000                                                                              12,000                                                                             5,200                                                                              7,800                                                                              4,700                                                                              8,600                                          30    4,500                                                                              8,600                                                                              5,100                                                                              6,000                                                                              6,000                                                                              8,200                                          31                             <10                                            32    5,000                                                                              9,000                                                                              3,800                                                                              6,200                                                                              1,400                                                                              4,600                                          33    7,900                                                                              22,000                                                                             11,000                                                                             20,000                                                                             12,000                                                                             14,000                                         34    5,200                                                                              14,000                                                                             150  200  48   170                                            35    4,600                                                                              7,400                                                                              4,200                                                                              5,100                                                                              300  590                                            38    330  12,000                                                                             4,600                                                                              11,000                                                                             8,500                                                                              16,000                                         39    1,700                                                                              12,000                                                                             4,500                                                                              8,400                                                                              4,500                                                                              11,000                                         40    1,600                                                                              11,500                                                                             3,200                                                                              5,000                                                                              210  230                                            41    3,200                                                                              17,000                                                                             3,600                                                                              8,000                                                                              8,000                                                                              8,600                                          42    300  380  270  450                                                      43    280  420  3,900                                                                              6,200                                                    44    6,600                                                                              9,300                                                                              5,000                                                                              6,200                                                    47    66   88   5,200                                                                              8,000                                                    48    60   85   380  400  500  550                                            49    150  850  160  240  240  270                                            50    34   140  36   58   20   36                                             51    14   120  62   68   17   30                                             52    340  660  260  530  200  270                                            53    1,900                                                                              7,200                                                                              2,600                                                                              3,200                                                                              1,800                                                                              2,700                                          54    140  220  62   140  170  200                                            55    5,200                                                                              14,000                                                                             4,000                                                                              5,900                                                                              1,900                                                                              2,000                                          56                                                                            57    290  1,000                                                                              140  260  860  1,000                                          58    110  260  22   64   400  430                                            59    3,500                                                                              10,500                                                                             1,600                                                                              2,100                                                                              1,500                                                                              2,600                                          60    3,500                                                                              4,800                                                                              2,600                                                                              3,200                                                                              510  630                                            61    170  1,100                                                                              6,300                                                                              6,900                                                                              550  750                                            62    25   340  38   68   92   140                                            63    36   170  19   63   23   35                                             64    180  1,200                                                                              32   42   280  480                                            65                                                                            66                                                                            67                                                                            73    940  1,400                                                              74    1,600                                                                              3,000                                                              75                                                                            76                                                                            __________________________________________________________________________    Sub-  Shigella  Shigella  Salmonella                                          strate                                                                              flexneri  sonnei    choleraesuis                                        Code No.                                                                            1 hr. (cpm)                                                                             1 hr. (cpm)                                                                             1 hr. (cpm)                                               Low  High Low  High Low  High                                           __________________________________________________________________________     7         <10       <10       <10                                            10    400  500  460  520  400  500                                            18         <10       <10  5,700                                                                              7,400                                          19         <10  1,000                                                                              2,100                                                                              500  1,600                                          20    41   60   5,300                                                                              6,600                                                                              1,800                                                                              4,400                                          21    20   230  180  190  <10  18                                             22    3,300                                                                              5,200                                                                              5,500                                                                              6,200                                                                              3,700                                                                              8,500                                          23         <10  60   72   37   84                                             24         <10  16   47        <10                                            25    70   170  90   180  800  1,100                                          26    <10  26   36   41   <10  21                                             27    3,700                                                                              7,600                                                                              5,900                                                                              6,700                                                                              2,200                                                                              6,500                                          28         <10       <10  <10  18                                             29    6,400                                                                              7,900                                                                              7,300                                                                              11,000                                                                             6,000                                                                              7,000                                          30    5,100                                                                              9,000                                                                              7,900                                                                              11,500                                                                             7,000                                                                              11,000                                         31                                                                            32    5,700                                                                              6,000                                                                              4,900                                                                              10,500                                                                             3,800                                                                              7,400                                          33    13,000                                                                             15,000                                                                             11,000                                                                             18,000                                                                             11,000                                                                             20,000                                         34    140  180  2,200                                                                              2,300                                                                              8,000                                                                              15,000                                         35    3,800                                                                              11,000                                                                             12,000                                                                             16,000                                                                             5,200                                                                              8,100                                          38    8,000                                                                              14,000                                                                             14,000                                                                             16,000                                                                             11,000                                                                             12,000                                         39    8,100                                                                              8,800                                                                              9,000                                                                              11,000                                                                             8,600                                                                              16,000                                         40    950  1,400                                                                              2,200                                                                              4,700                                                                              7,200                                                                              8,100                                          41    5,800                                                                              6,400                                                                              7,800                                                                              9,500                                                                              11,000                                                                             12,000                                         42    180  190  660  880  400  460                                            43    6,000                                                                              6,500                                                                              7,200                                                                              8,400                                                                              8,900                                                                              14,000                                         44    6,300                                                                              6,600                                                                              7,600                                                                              12,000                                                                             6,100                                                                              8,000                                          47    9,500                                                                              10,000         12,000                                                                             19,000                                         48    450  530  410  820  430  540                                            49    250  620  75   160  78   120                                            50    330  420  32   140  54   71                                             51    40   60   13   82   15   50                                             52    880  3,300                                                                              580  1,800                                                                              270  800                                            53    1,900                                                                              2,700                                                                              2,000                                                                              5,600                                                                              2,300                                                                              3,300                                          54    130  160  95   230  110  140                                            55    2,500                                                                              4,700                                                                              5,400                                                                              8,900                                                                              7,000                                                                              8,300                                          56              16   82   51   53                                             57    450  750  90   270  38   140                                            58    100  160  68   120  30   61                                             59    1,400                                                                              1,800                                                                              540  3,600                                                                              270  900                                            60    130  140  74   170  35   50                                             61    1,200                                                                              1,300                                                                              2,300                                                                              4,400                                                                              95   140                                            62    27   55   24   98   13   41                                             63    12   38   10   36   <10  35                                             64    260  320  430  1,100                                                                              85   120                                            65                                                                            66                                                                            67                                                                            73                                                                            74                                                                            75                                                                            76                                                                            __________________________________________________________________________    Sub-  Salmonella                                                                              Salmonella                                                                              Edwardsiella                                        strate                                                                              pullorum  galinarum tarda                                               Code No.                                                                            1 hr. (cpm)                                                                             1 hr. (cpm)                                                                             1 hr. (cpm)                                               Low  High Low  High Low  High                                           __________________________________________________________________________     7         <10       <10       <10                                            10    130  290  120  190  450  600                                            18    8,100                                                                              9,300                                                                              800  1,100                                                                              2,800                                                                              11,000                                         19    41   98   420  500  400  1,500                                          20    21   65   600  700  2,100                                                                              4,700                                          21    260  280  350  470  310  470                                            22    11,000                                                                             12,000                                                                             8,000                                                                              9,000                                                                              7,000                                                                              9,500                                          23    15   75   54   92   1,600                                                                              2,000                                          24    <10  31   <10  27   <10  70                                             25    110  330  160  180  22   75                                             26    <10  39   60   150  <10  56                                             27    850  1,200                                                                              9,500                                                                              11,000                                                                             260  430                                            28    <10  30   <10  18        <10                                            29    1,100                                                                              1,400                                                                              1,100                                                                              4,600                                                                              2,200                                                                              4,200                                          30    1,300                                                                              1,700                                                                              11,000                                                                             14,000                                                                             170  760                                            31                                                                            32    1,100                                                                              1,400                                                                              10,000                                                                             18,000                                                                             840  3,700                                          33    300  380  880  950  560  13,000                                         34    910  1,100                                                                              8,200                                                                              10,500                                                                             9,000                                                                              12,000                                         35    680  780  350  420  850  5,200                                          38    1,200                                                                              1,700                                                                              9,700                                                                              13,000                                                                             11,500                                                                             14,000                                         39    1,200                                                                              1,700                                                                              5,300                                                                              13,000                                                                             7,500                                                                              12,000                                         40    230  430  4,100                                                                              8,100                                                                              2,900                                                                              12,000                                         41    1,000                                                                              1,700                                                                              10,000                                                                             17,000                                                                             9,000                                                                              14,000                                         42    620  690  310  460  3,400                                                                              4,500                                          43    710  1,300                                                                              1,500                                                                              2,700                                                                              7,000                                                                              8,000                                          44    1,300                                                                              1,500                                                                              12,000                                                                             15,000                                                                             9,500                                                                              11,000                                         47                                                                            48              380  2,800                                                                              4,500                                                                              5,600                                          49                        580  650                                            50                        81   95                                             51                        51   71                                             52                        210  250                                            53                        1,100                                                                              1,200                                          54                        55   74                                             55                        1,600                                                                              2,600                                          56                                                                            57                        170  230                                            58                        35   95                                             59                        150  210                                            60                        52   58                                             61                        73   96                                             62                        42   48                                             63                        12   42                                             64                        5,800                                                                              9,900                                          65                                                                            66                                                                            67                                                                            73                                                                            74                                                                            75                                                                            76                                                                            __________________________________________________________________________    Sub-  Arizona   Citrobacter                                                                             Klebsiella                                          strate          freuendii pneumonia                                           Code No.                                                                            1 hr. (cpm)                                                                             1 hr. (cpm)                                                                             1 hr. (cpm)                                               Low  High Low  High Low  High                                           __________________________________________________________________________     7    <10  35        <10  12   37                                             10    110  520  150  600  350  1,200                                          18    350  5,300                                                                              4,800                                                                              12,000                                                                             510  1,400                                          19    95   8,200                                                                              5,000                                                                              16,000                                                                             1,900                                                                              5,400                                          20    280  2,900                                                                              1,500                                                                              5,900                                                                              1,300                                                                              3,700                                          21    80   330  62   210  2,100                                                                              5,000                                          22    380  7,200                                                                              2,100                                                                              6,000                                                                              4,100                                                                              6,200                                          23    160  4,200                                                                              950  4,200                                                                              850  1,400                                          24    <10  45        <10       <10                                            25    70   350  130  420  120  720                                            26    15   450  210  950  15   92                                             27    400  4,800                                                                              2,700                                                                              6,200                                                                              3,200                                                                              13,000                                         28    <10  35        <10  <10  36                                             29    500  8,500                                                                              4,700                                                                              8,200                                                                              2,100                                                                              7,500                                          30    1,800                                                                              8,500                                                                              4,100                                                                              9,200                                                                              3,500                                                                              7,500                                          31                                                                            32    1,300                                                                              8,500                                                                              5,800                                                                              11,000                                                                             4,800                                                                              8,500                                          33    3,300                                                                              24,000                                                                             8,000                                                                              20,000                                                                             8,800                                                                              15,000                                         34    290  5,500                                                                              5,100                                                                              9,600                                                                              7,200                                                                              9,000                                          35    1,900                                                                              15,000                                                                             3,100                                                                              13,000                                                                             850  4,000                                          38    610  9,500                                                                              1,900                                                                              14,000                                                                             4,000                                                                              16,000                                         39    1,400                                                                              12,000                                                                             5,800                                                                              8,800                                                                              7,500                                                                              12,000                                         40    250  12,000         3,400                                                                              9,000                                          41    1,500                                                                              8,000          4,500                                                                              8,000                                          42                        580  750                                            43                        6,500                                                                              9,000                                          44                        6,000                                                                              7,000                                          47    6,500                                                                              8,000                                                                              5,800                                                                              6,200                                                                              8,800                                                                              15,000                                         48    4,000                                                                              5,500                                                                              2,000                                                                              3,400                                                                              3,200                                                                              12,000                                         49    380  700  250  450  380  18,000                                         50    110  600  88   150  310  950                                            51    14   52   <10  32   <10  24                                             52    800  1,500                                                                              75   27   900  1,200                                          53    2,000                                                                              5,500                                                                              2,100                                                                              2,600                                                                              3,800                                                                              5,700                                          54    320  1,400                                                                              620  2,600                                                                              420  1,200                                          55    6,000                                                                              10,000                                                                             3,100                                                                              5,800                                                                              2,400                                                                              9,400                                          56                                                                            57    410  620  170  320  4,200                                                                              1,800                                          58    850  550  40   100  32   520                                            59    3,200                                                                              4,300                                                                              2,100                                                                              3,200                                                                              2,100                                                                              4,700                                          60    3,200                                                                              7,000                                                                              3,200                                                                              6,800                                                                              6,000                                                                              11,000                                         61    2,300                                                                              6,200                                                                              5,200                                                                              8,500                                                                              1,900                                                                              5,500                                          62    38   450  51   330  100  800                                            63    18   29   16   72   240  550                                            64    120  390  450  2,700                                                                              92   170                                            65                                                                            66                                                                            67                                                                            73                                                                            74                                                                            75                                                                            76                                                                            __________________________________________________________________________    Sub-  Klebsiella                                                                              Klebsiella                                                                              Enterobacter                                        strate                                                                              ozaenae   rhinoschleromatis                                                                       cloacae                                             Code No.                                                                            1 hr. (cpm)                                                                             1 hr. (cpm)                                                                             1 hr. (cpm)                                               Low  High Low  High Low  High                                           __________________________________________________________________________     7         <10       <10       <10                                            10    310  330  32   42   380  420                                            18         <10  <10  16   4,100                                                                              6,200                                          19    3,200                                                                              5,100                                                                              34   65   4,000                                                                              5,500                                          20    500  980  2,100                                                                              2,500                                                                              2,000                                                                              2,300                                          21    81   170  180  230  3,400                                                                              5,000                                          22    4,000                                                                              4,600                                                                              4,700                                                                              6,500                                                                              5,500                                                                              8,500                                          23    1,100                                                                              2,300                                                                              140  160  410  1,400                                          24         <10  <10  27   <10  16                                             25    45   68   140  210  90   420                                            26    34   36   50   75   <10  63                                             27    1,200                                                                              1,900                                                                              4,500                                                                              5,200                                                                              3,500                                                                              7,500                                          28    160  950       <10  32   75                                             29    4,800                                                                              5,200                                                                              3,200                                                                              3,700                                                                              4,600                                                                              6,500                                          30    4,500                                                                              9,500                                                                              200  250  5,500                                                                              7,500                                          31                                                                            32    5,500                                                                              7,400                                                                              5,000                                                                              9,000                                                                              5,500                                                                              7,500                                          33    12,000                                                                             22,000                                                                             5,000                                                                              15,000                                                                             10,000                                                                             14,000                                         34    9,000                                                                              12,500                                                                             9,200                                                                              12,000                                                                             5,500                                                                              8,000                                          35    12,000                                                                             15,000                                                                             6,200                                                                              7,500                                                                              6,200                                                                              7,000                                          38    11,000                                                                             12,000                                                                             9,500                                                                              11,000                                                                             7,800                                                                              12,000                                         39    10,000                                                                             12,000                                                                             7,500                                                                              14,000                                                                             8,000                                                                              9,100                                          40    7,500                                                                              9,500                                                                              6,000                                                                              6,500                                                                              6,100                                                                              6,700                                          41    7,000                                                                              12,000                                                                             8,800                                                                              12,000                                                                             10,000                                                                             11,000                                         42    1,600                                                                              2,700                                                                              340  410  2,400                                                                              2,600                                          43    5,100                                                                              9,500                                                                              6,200                                                                              10,000                                                                             8,800                                                                              9,000                                          44    6,600                                                                              11,000                                                                             8,000                                                                              9,000                                                                              7,000                                                                              7,500                                          47                        7,000                                                                              11,000                                         48    5,200                                                                              10,000                                                                             4,100                                                                              4,800                                                                              3,200                                                                              7,800                                          49    410  730  300  740  310  1,200                                          50    410  800  45   170  1,200                                                                              6,500                                          51    35   65   32   85   <10  700                                            52    320  590  1,100                                                                              1,500                                                                              480  1,500                                          53    520  830  1,800                                                                              2,800                                                                              3,000                                                                              9,500                                          54    150  300  58   120  200  8,100                                          55    650  950  4,500                                                                              6,400                                                                              4,200                                                                              11,000                                         56    180  220                                                                57    310  420  130  400  3,300                                                                              28,000                                         58    100  170  48   130  780  1,300                                          59    2,200                                                                              5,700                                                                              3,300                                                                              5,600                                                                              1,700                                                                              4,100                                          60    5,800                                                                              7,000                                                                              35   150  2,100                                                                              12,000                                         61    1,200                                                                              1,800                                                                              1,500                                                                              2,000                                                                              2,300                                                                              4,900                                          62    60   180  58   92   2,100                                                                              7,200                                          63    33   65   40   82   780  2,200                                          64    280  350  110  200  320  580                                            65                                                                            66                                                                            67                                                                            73                                                                            74                                                                            75                                                                            76                                                                            __________________________________________________________________________    Sub-  Enterobacter                                                                            Enterobacter                                                                            Enterobacter                                        strate                                                                              aerogenes hafnia    liquefaciens                                        Code No.                                                                            1 hr. (cpm)                                                                             1 hr. (cpm)                                                                             1 hr. (cpm)                                               Low  High Low  High Low  High                                           __________________________________________________________________________     7         <10       <10       <10                                            10    170  510  1,400                                                                              2,900                                                                              180  500                                            18    5,800                                                                              12,000                                                                             100  520  2,900                                                                              8,000                                          19    4,000                                                                              11,000                                                                             780  1,100                                                                              4,000                                                                              7,800                                          20    1,900                                                                              2,100                                                                              1,500                                                                              1,800                                                                              2,400                                                                              3,800                                          21    2,100                                                                              2,700                                                                              88   220  150  230                                            22    5,100                                                                              6,600                                                                              550  680  5,800                                                                              7,000                                          23    3,100                                                                              5,000                                                                              920  1,400                                                                              1,100                                                                              3,700                                          24    <10  20        <10  <10  61                                             25    13   28   <10  17   160  500                                            26    11   56   <10  52   51   140                                            27    3,200                                                                              7,500                                                                              20   1,200                                                                              1,900                                                                              4,400                                          28    550  600  450  700  320  1,300                                          29    3,000                                                                              9,000                                                                              7,000                                                                              8,000                                                                              5,500                                                                              9,500                                          30    4,700                                                                              9,500                                                                              4,100                                                                              8,200                                                                              5,500                                                                              9,500                                          31                                                                            32    6,000                                                                              11,000                                                                             4,800                                                                              7,200                                                                              7,500                                                                              12,000                                         33    11,000                                                                             19,000                                                                             8,500                                                                              17,000                                                                             13,000                                                                             18,000                                         34    4,800                                                                              9,800                                                                              4,700                                                                              5,200                                                                              5,300                                                                              12,000                                         35    2,400                                                                              11,000                                                                             2,200                                                                              8,500                                                                              2,500                                                                              4,100                                          38    5,400                                                                              10,000                                                                             8,100                                                                              11,000                                                                             7,100                                                                              13,000                                         39    5,500                                                                              11,000                                                                             6,800                                                                              8,000                                                                              8,200                                                                              11,000                                         40    10,000                                                                             11,000                                                                             9,000                                                                              9,500                                                                              5,000                                                                              9,500                                          41    11,000                                                                             12,000                                                                             4,400                                                                              7,500                                                                              8,000                                                                              12,000                                         42              5,100                                                                              5,500                                                                              4,500                                                                              6,200                                          43              5,100                                                                              5,500                                                                              6,800                                                                              9,500                                          44              7,200                                                                              7,500                                                                              7,400                                                                              9,000                                          47              9,900                                                                              10,500                                                   48    3,800                                                                              5,600                                                                              3,200                                                                              7,800                                                                              506  900                                            49    880  960  4,800                                                                              9,600                                                                              560  950                                            50    210  460  1,200                                                                              5,300                                                                              460  730                                            51    23   40   30   52   31   67                                             52    210  480  320  1,100                                                                              310  350                                            53    260  380  2,100                                                                              3,500                                                                              1,200                                                                              1,700                                          54    320  400  600  900  300  450                                            55    9,500                                                                              11,000                                                                             4,400                                                                              9,500                                                                              4,200                                                                              7,000                                          56    25   36             320  420                                            57    110  220  1,800                                                                              3,000                                                                              230  430                                            58    43   73   130  590  60   180                                            59    2,100                                                                              3,600                                                                              4,500                                                                              8,500                                                                              3,200                                                                              9,500                                          60    2,500                                                                              4,500                                                                              60   3,500                                                                              2,700                                                                              5,200                                          61    950  2,700                                                                              950  3,000                                                                              750  1,200                                          62    15   59   50   390  42   580                                            63    12   31   120  270  31   280                                            64    550  620  380  1,100                                                                              410  490                                            65                                                                            66                                                                            67                                                                            73                                                                            74                                                                            75                                                                            76                                                                            __________________________________________________________________________    Sub-  Pectobacterium                                                                          Serratia  Proteus                                             strate                                                                              carotovarum                                                                             marcescens                                                                              vulgaris                                            Code No.                                                                            1 hr. (cpm)                                                                             1 hr. (cpm)                                                                             1 hr. (cpm)                                               Low  High Low  High Low  High                                           __________________________________________________________________________     7         <10       <10  220  9,000                                          10    850  950  230  420  95   350                                            18    8,500                                                                              13,000                                                                             4,300                                                                              5,100                                                                              350  7,500                                          19    95   140  3,500                                                                              4,000                                                                              18   85                                             20    65   120  3,500                                                                              4,000                                                                              310  1,600                                          21    6,100                                                                              6,800                                                                              3,500                                                                              4,800                                                                              2,200                                                                              8,500                                          22    8,000                                                                              9,000                                                                              6,300                                                                              8,200                                                                              3,800                                                                              7,800                                          23    <10  22   850  1,800                                                                              <10  58                                             24    28   88        <10  <10  65                                             25    180  270  160  330  <10  110                                            26    60   70        <10  <10  220                                            27    5,500                                                                              8,000                                                                              3,500                                                                              8,000                                                                              <10  60                                             28    < 10 32   150  600  <10  45                                             29    650  700  6,500                                                                              7,000                                                                              310  1,700                                          30    900  1,000                                                                              5,000                                                                              7,000                                                                              290  900                                            31                                                                            32    620  920  5,800                                                                              8,000                                                                              2,500                                                                              9,500                                          33    12,000                                                                             13,000                                                                             11,000                                                                             17,000                                                                             32   250                                            34    9,000                                                                              11,000                                                                             5,500                                                                              12,000                                                                             1,600                                                                              8,500                                          35    1,800                                                                              2,600                                                                              2,200                                                                              6,800                                                                              1,100                                                                              6,200                                          38    7,000                                                                              8,000                                                                              3,100                                                                              7,500                                                                              8,500                                                                              17,000                                         39    4,800                                                                              6,200                                                                              6,500                                                                              10,500                                                                             7,200                                                                              17,000                                         40    2,300                                                                              2,600                                                                              5,500                                                                              6,000                                                                              3,800                                                                              13,000                                         41    12,000                                                                             18,000                                                                             8,500                                                                              13,000                                                                             4,500                                                                              16,000                                         42    500  600  5,800                                                                              6,200                                                    43    500  600  8,000                                                                              9,000                                                    44    11,000                                                                             14,000                                                                             8,000                                                                              9,000                                                    47              9,000                                                                              10,000                                                                             5,000                                                                              6,000                                          48    1,100                                                                              3,100                                                                              4,800                                                                              9,800                                                                              320  850                                            49    280  390  3,909                                                                              9,200                                                                              180  700                                            50    78   120  1,800                                                                              2,700                                                                              45   550                                            51    35   65   <10  52   <10  16                                             52    950  2,700                                                                              220  800  50   250                                            53    1,600                                                                              3,300                                                                              380  600  250  650                                            54    70   100  260  550  550  360                                            55    2,800                                                                              3,100                                                                              3,200                                                                              9,000                                                                              11,000                                                                             14,000                                         56    250  460                                                                57    150  220  1,100                                                                              2,800                                                                              500  4,000                                          58    55   120  40   100  85   200                                            59    7,500                                                                              9,000                                                                              5,700                                                                              9,500                                                                              1,800                                                                              3,900                                          60    850  1,300                                                                              9,200                                                                              20,000                                                                             700  1,500                                          61    170  180  1,100                                                                              5,000                                                                              120  250                                            62    35   64   41   180  55   1,800                                          63    25   39   52   350  <10  45                                             64              800  6,000                                                                              85   370                                            65                        500  700                                            66                                                                            67                                                                            73                                                                            74                                                                            75                                                                            76                                                                            __________________________________________________________________________    Sub-  Proteus   Proteus   Proteus                                             strate                                                                              mirabilis rettgeri  morganii                                            Code No.                                                                            1 hr. (cpm)                                                                             1 hr. (cpm)                                                                             1 hr. (cpm)                                               Low  High Low  High Low  High                                           __________________________________________________________________________     7    150  290  550  850  320  2,100                                          10    310  700  210  270  350  620                                            18    630  750  5,000                                                                              6,000                                                                              510  1,400                                          19    30   280       <10  19   51                                             20    78   120  20   45   35   52                                             21    320  360       <10  <10  20                                             22    5,200                                                                              5,800                                                                              4,000                                                                              6,000                                                                              3,900                                                                              7,100                                          23    <10  15        <10       <10                                            24    18   52        <10       <10                                            25    48   63   <10  21   16   28                                             26    58   62   <10  26   <10  62                                             27    <10  140  220  410  240  260                                            28         <10  <10  61        <10                                            29    6,500                                                                              12,000                                                                             300  350  1,900                                                                              2,000                                          30    7,500                                                                              10,500                                                                             4,000                                                                              5,500                                                                              3,100                                                                              5,100                                          31                                                                            32    7,500                                                                              9,000                                                                              950  1,900                                                                              1,200                                                                              1,400                                          33    13,000                                                                             19,000                                                                             12,000                                                                             13,000                                                                             12,000                                                                             14,000                                         34    4,000                                                                              7,100                                                                              5,600                                                                              7,400                                                                              420  510                                            35    3,500                                                                              5,500                                                                              16   22   5,200                                                                              7,200                                          38    8,500                                                                              9,500                                                                              6,600                                                                              9,000                                                                              3,700                                                                              5,200                                          39    6,000                                                                              8,000                                                                              6,000                                                                              7,000                                                                              850  3,300                                          40    1,400                                                                              1,500                                                                              4,000                                                                              6,500                                                                              5,800                                                                              6,200                                          41    9,000                                                                              21,000                                                                             8,000                                                                              9,800                                                                              5,800                                                                              7,000                                          42    850  900  1,500                                                                              2,500                                                                              700  2,000                                          43    9,500                                                                              10,000                                                                             2,500                                                                              3,500                                                                              4,000                                                                              4,800                                          44    8,000                                                                              9,000                                                                              8,000                                                                              8,800                                                                              6,500                                                                              7,500                                          47              9,500                                                                              11,000                                                                             8,000                                                                              9,000                                          48    650  780  1,100                                                                              4,700                                                                              280  450                                            49    65   150  450  1,500                                                                              75   270                                            50    21   90   240  850  120  480                                            51    <10  21   51   520  12   33                                             52    13   280  210  700  120  530                                            53    230  1,600                                                                              2,900                                                                              4,100                                                                              1,400                                                                              2,300                                          54    140  580  160  1,600                                                                              140  3,100                                          55    5,000                                                                              11,000                                                                             6,500                                                                              9,200                                                                              5,000                                                                              8,800                                          56    2,400                                                                              2,800                                                                              60   1,600                                                                              1,000                                                                              1,600                                          57    230  310  85   4,500                                                                              220  580                                            58    16   110  100  1,600                                                                              <10  160                                            59    1,900                                                                              3,300                                                                              1,700                                                                              4,000                                                                              2,200                                                                              5,000                                          60    12   85   380  2,600                                                                              270  1,700                                          61    1,800                                                                              8,500                                                                              600  960  75   720                                            62    900  2,600                                                                              96   2,100                                                                              16   1,300                                          63    1,100                                                                              2,900                                                                              35   550  <10  920                                            64    650  1,900                                                                              610  750  34   750                                            65                                                                            66                                                                            67                                                                            73                                                                            74                                                                            75                                                                            76                                                                            __________________________________________________________________________    Sub-  Providence                                                                              Providence                                                                              Pseudomonas                                         strate                                                                              stuartii  alcalifacens                                                                            aernginosa                                          Code No.                                                                            1 hr. (cpm)                                                                             1 hr. (cpm)                                                                             1 hr. (cpm)                                               Low  High Low  High Low  High                                           __________________________________________________________________________     7                   <10       <10                                            10              350  420  370  390                                            18              4,800                                                                              6,200                                                                              1,600                                                                              3,800                                          19              40   75   5,000                                                                              6,600                                          20              45   95   100  1,600                                          21              65   700  210  230                                            22              2,500                                                                              4,500                                                                              1,900                                                                              2,700                                          23                   <10  2,700                                                                              4,800                                          24                   <10  <10  28                                             25              31   80   90   100                                            26                   <10  22   56                                             27              250  300  65   8,500                                          28              <10  18   3,500                                                                              4,000                                          29              2,300                                                                              7,600                                                                              5,400                                                                              6,600                                          30              1,100                                                                              3,600                                                                              8,000                                                                              9,400                                          31                                                                            32              1,200                                                                              6,200                                                                              8,000                                                                              12,000                                         33              9,200                                                                              11,000                                                                             5,000                                                                              9,400                                          34              5,700                                                                              14,000                                                                             6,100                                                                              11,000                                         35              48   140  4,500                                                                              5,500                                          38              12,000                                                                             14,000                                                                             5,000                                                                              11,000                                         39              8,500                                                                              11,000                                                                             5,800                                                                              10,500                                         40              6,300                                                                              16,000                                                                             880  2,200                                          41              13,000                                                                             15,000                                                                             8,200                                                                              11,000                                         42                        7,600                                                                              8,300                                          43                        5,600                                                                              6,000                                          44              7,500                                                                              8,000                                                                              8,800                                                                              9,200                                          47              14,000                                                                             15,000                                                                             8,000                                                                              10,000                                         48              4,400                                                                              6,200                                                                              1,200                                                                              1,600                                          49    750  900  520  1,000                                                                              1,300                                                                              1,600                                          50    120  130  160  390  1,900                                                                              2,200                                          51    180  220  210  370  28   58                                             52    380  450  170  1,100                                                                              100  150                                            53    2,800                                                                              3,200                                                                              5,400                                                                              16,000                                                                             220  280                                            54    400  450  280  3,200                                                                              210  240                                            55    6,700                                                                              7,400                                                                              3,000                                                                              6,200                                                                              3,100                                                                              3,700                                          56                                                                            57    260  480  1,300                                                                              5,600                                                                              2,800                                                                              4,000                                          58    110  180  11,000                                                                             22,000                                                                             210  250                                            59    5,800                                                                              6,200                                                                              11   22   1,300                                                                              1,400                                          60    120  160  350  740  5,800                                                                              6,200                                          61    570  950  16   280  1,500                                                                              4,000                                          62    140  180  80   110  90   230                                            63    62   90   26   300  700  2,200                                          64    140  180  120  180  420  720                                            65                                                                            66                                                                            67                                                                            73                                                                            74                                                                            75                                                                            76                                                                            __________________________________________________________________________

The following example illustrates the procedure for the identificationof pathogenic bacteria by the radioisotopic identification technique ofthis invention:

EXAMPLE

A sample of a human stool is received in the laboratory. The history ofthe patient indicates the possibility of a Salmonella infection. Thesample material is streaked onto agar plates including Eosine MethyleneBlue Agar, MacConkey Agar, Blood agar, Bismuth Sulfite Agar, SalmonellaShigella agar and Selenite-F Broth. Each inoculated media is thenincubated at 35°C. overnight. The next day, the plates are observed andsome are found to show bacterial colonies which have the appearance ofsalmonella. Several typical colonies are picked from the agar plateswith a swab and suspended in a saline solution. The cell density isadjusted to an optical density of approximately 1 as determined by aspectrophotometer. About 0.05 ml. of the cell suspension is addeddropwise to each well in a microculture tray. Each well on the traycontains 0.05 microcuries of a different one of the following ¹⁴ Clabeled substrates identified by the code numbers set forth in Table 2:7, 18, 19, 20, 21, 22, 23, 24, 25, 27, 28, 30, 33, 34, 35, 40, 42, 43,44, 47, 50, 53, 55, 57, 58, 59, 60, 61, 62, 63. The inoculatedmicroculture tray is covered with a sheet impregnated with Ba(OH)₂, theportion of the cover overlaying each well being coded so as to identifythe substrate in that well. The tray is incubated for one hour and theBa(OH)₂ cover is removed and dried under a heat lamp. The microculturetray is discarded into a receptacle for containing radioactive wastematerials. The dried Ba(OH)₂ containing pad is measured forradioactivity and the counts per minute corresponding to each ¹⁴ Clabeled substrate is automatically recorded. There is thereby obtained aradiorespirometric profile of the unknown sample material. This profileis compared to standard radiorespirometric profiles of the organisms setforth in Table 1. Comparison is performed by means of a computer whichtests the goodness of fit and statistical reliability. Theradiorespirometric profile of the unknown organism is found to compareto the standard radiorespirometric profile of the organism Salmonellatyphi.

The steps performed on the first day, i.e., the streaking of the samplematerial on the agar plates followed by overnight incubation, arerequired by either the conventional or the radiorespirometricidentification technique of this invention. However, the steps performedon the second day for the radiorespirometric technique do not requirehighly experienced personnel which are required by conventionalidentification techniques since the interpretation of results is notrequired--i.e., the identification is made automatically. Moreover, thetime required to perform the steps on the second day of theradiorespirometric identification technique is about five minutesmanipulation time and approximately 1 hour and 10 minutes total elapsedtime which is considerably less than the day or more required byconventional identification techniques.

There will now be described apparatus for collecting and analyzing datarelating to a plurality of samples of organisms or the like. A suitableincubation unit assembly for use with an automatic analyzing system isshown in FIGS. 10 and 11 and inclues a culture tray 20 having aplurality of generally hemispherical depressions 21 spaced in a regulararray of columns and rows throughout the body of the tray. While thetray can be produced as a solid, self-supporting unit similar to thatshown in FIGS. 2-4, a relatively inexpensive, disposable and yet equallysuitable apparatus can be produced by vacuum forming the tray from arelatively thin sheet of a thermo-formable plastic material. The tray isthen designed for use with a reusable support body such as thatindicated generally at 23, the support body being relatively thick andrigid and having a plurality of wells 24 spaced in the same array as intray 20. The upper surface of body 23 is provided with an alignment pin25 which is designed to pass through an opening 26 provided in tray 20so that the apparatus will be in proper orientation as well asalignment. The lower surface of body 23 is provided with a plurality ofdownwardly protruding parallel ribs 27 which extend substantially thelength of body 23 and are intended to perform a spacing function duringincubation, as will be described.

The apparatus also includes a collection material cover unit indicatedgenerally at 29 which includes a backing member 30 and an absorbentmaterial sheet 31 which constitutes the CO₂ collector previouslydescribed. The backing member 30 is a relatively stiff plastic memberwhich is generally planar in configuration but which is provided withdownwardly extending edges 33 and 34 which serve as stiffeners and asspacers in the analysis system to be described.

As seen in FIG. 11, when the materials are inserted into depressions 21and the collector material in cover 29 is placed thereon, the assemblyconstitutes a relatively thin, easily manageable structure in which thecover forms a tight seal over each of the depressions, isolating eachsuch depression from each other depression and from the externalatmosphere.

During the incubation period a plurality of such assemblies can bestacked one upon the other as shown in FIG. 12. In that figure, eachassembly is spaced from each lower assembly by the downwardly extendingribs 27 to permit the passage of air, thereby maintaining a uniformtemperature environment for each incubation unit, regardless of itsposition in the stack. A weight 35 is placed on the cover 29 of thetopmost unit to be sure that the cover of that unit, and all lowerunits, is tightly covering the depressions in the respective trays 20.Each body 23 can be provided with a legend indicating the time when theincubation for that specific tray is to be terminated so that it can beremoved at the appropriate time for further processing andidentification procedures.

It will also be observed that the ribs 27, being rather uniformlydisposed across the width of each body 23, apply rather uniform pressureto the cover 29 of the unit below it, and that, furthermore, the ribsare disposed beneath rows of wells so that the cover of the unit onwhich the ribs press is pressed against the well openings, i.e., atexactly the position where pressure is most needed.

As previously described, after the incubation period has transpired thecovers are removed and analyzed for level of radioactive material in thespecific regions covering the wells. A particularly advantageousapparatus for accomplishing this analysis will now be described.

It will be observed, preliminarily, that a cover such as that describedfor use with the apparatus of FIGS. 10-12 will be that as shown in FIG.13 wherein a cover 29 is provided with an absorbent layer 31, this beingseen from the undersurface or in an inverted position in FIG. 13. Thecover is also provided with an alignment hole 28 for alignment with thebodies supporting the material to be analyzed.

The apparatus to be described, however, is also capable of handling andanalyzing materials which have been exposed in a different fashion,specifically in the form of separable pads which can be exposed to aculture and then placed in the cover. For this purpose, a cover of thetype shown in FIG. 14 is provided, this constituting a relatively rigidshallow U-shaped tray 40 which is provided with a plurality ofdepressions 41 to receive pads of absorbent material 42 which are to beinvestigated. The depressions 41 are shallow and circular and arearranged in regular rows and columns, preferably in the same kind ofarray employed with depression 24 in body 23 or wells 21 in the culturetray 20 described with reference to FIGS. 10 and 11. Using the samearray permits analysis with the same equipment.

An automatic apparatus for analyzing the material on the covers is shownin FIG. 15 in a general and schematic fashion. A plurality of covers 45are stacked one upon the other immediately above a continuous conveyorbelt 46. The covers are maintained in an aligned position possibly asshown in FIG. 15 between upstanding posts 47. The covers are maintainedin spaced relationship by the edges thereof, as shown at 33 and 34 inFIG. 11. It will be observed that the covers can be of the form shown inFIG. 13 or of the form shown in FIG. 14.

A source of hot air 48 is provided in the vicinity of the covers to passair between the covers, thereby rendering them in a dry state.

The conveyor 46 is provided with sets of upwardly extending lugs inpairs as shown at 50, 51 and 52. The pairs of lugs are spaced apart bydistances greater than the length of a cover. When a set of lugs such as51 arrives at the bottom of one of covers 45, the lugs engage the rearedge of the cover and move it along with the belt past the analyzingapparatus, after which the cover is dropped or removed from the end ofthe conveyor and either discarded, returned to the input end for arepeat run, or cleaned and prepared for further use.

The analyzing apparatus which is broadly illustrated in FIG. 15 includesa plurality of sensitive detectors indicated generally at 55, thesedetectors being selected for sensitivity to the specific kind ofradioactivity to which the regions of absorbent material 31, or pads 42have been exposed. In particular, it is contemplated that Geiger-Mullerdetectors are to be employed and that the number of such tubes is equalto the number of regions in the width of the covers to be inspected.Additionally, the tubes are in the same array as one row of thoseregions and as one row of wells 21 in sample tray 20.

Detectors 55 produce electrical signals representative of the counts ofradioactive particles emitted from the specific regions underexamination and those signals are then transmitted over conductorsindicated generally at 56 to a data processing and display unit 57 whichwill be described in greater detail. Generally speaking, however, thedata processing apparatus receives the count information from thedetectors, compares it with a library of known information describingpreviously identified material and produces a display, such as aprintout, identifying the detected material as one previously identifiedor indicating that no such valid comparison has been made.

In order for the process of detection and analysis to be properlyconducted, it is desirable that each set of regions be moved to aposition directly adjacent the detectors 55, be retained in thatposition for a predetermined interval of time and then moved. It is alsodesirable that the counters be controlled to read and reset at thebeginning and end of each such count, transmitting the count at thereset time. The control of conveyor 46 and the counter is synchronizedand performed by a control unit 58 which will also be described ingreater detail. Control unit provides the energization of a drive motor59 and a drive motor 60 which provide the motivating force for conveyorbelt 46.

FIG. 16 shows, in a block schematic diagram form, an apparatus usable inthe data processing device referred to in FIG. 15 and used to performthe automatic method of the invention in conjunction with the conveyorand analysis apparatus disclosed therein. A plurality of particledetectors are indicated generally at 55, each of these being connectedto a source of D.C. voltage which is conventionally required, thevoltage being a relatively high voltage in the case of the usualGeiger-Muller tubes. One such tube and the apparatus used therewith willbe described in detail. The remaining tubes are provided withsubstantially identical processing channels.

Each tube includes an envelope 65 which contains an ionizable gas, theenvelope surrounding an electrode 66 connected to one terminal of theD.C. source 67. The other terminal of the source is connected to anelectrode 68 which extends into the ionizable gas and detects theavalanche ionization activity indicative of the exposure of the deviceto a particle. Each such activity generates a pulse electrical outputwhich is coupled through a capacitor 69 to the input of a conventionalamplifier 70 which can be employed to strengthen the signal and, ifdesired, to perform a shaping function.

The output of amplifier 70 is connected through an AND gate 71 to theinput of a counter 72 which receives the pulses gated through fromamplifier 70 and accumulates a count thereof. The output of the countercan be transferred to a buffer storage unit 74 for temporary storage ofthe count, rendering it available to further processing apparatus.

A drive control unit 58 operates in conjunction with a sequence controlor multiplying apparatus 75 and includes timing devices to activate thevarious portions of the system in proper order. As described previouslyin connection with the method of the present invention, it is desirableto accumulate a count representative of the mean rate of emission ofparticles from each region of the absorbent material being investigated.Thus, it is necessary to control the interval of time during which eachtube is exposed to each specific region from which a radiation count isto be obtained. This interval is controlled by AND gate 71 and the inputto the AND gate on conductor 73, which input is provided from controlcircuitry within control unit 58. That control unit opens gate 71,permitting pulses to be delivered to the counters only during thepredetermined interval.

Control unit 58 also provides control signals to the sequence selectoror multiplexor 75 to permit the sequence selector to transferaccumulated count information from the buffer 74 and the similar buffersassociated with the other Geiger-Muller tubes in the sensor array, thecounts being individually provided to a computer 76 which furtherprocesses the information and provides a usable result. The function ofthe sequence selector is to provide the accumulated counts in anestablished, ordered sequence so that they can be further analyzed. Ifnecessary, the count selected by the sequence selector can be providedto a translator 77, the function of which is recognizable by thecomputer. The transmitted information is then inserted into a store 78within the computer and it is compared with previously storedinformation contained in a data file library 79, the comparison beingaccomplished in a word comparator 80. The results of the comparison,including the identification of all matches occurring between the datain store 78 and library 79, is printed or otherwise displayed in a readout unit 81 of conventional nature. The comparison operation and thecontrol of the library scan and coordination of these operations withthe sequencer are accomplished by an internal control unit 82 whichprovides control signals and receives accomplishment feedback signalsfrom the comparison unit, the library and control unit 58.

As previously indicated, the analysis system described herein is capableof performing analysis of various types. In order for the dataprocessing apparatus to appropriately function, it is necessary thatreference be made, for example, to the appropriate library, and it isfurther necessary for the control and comparison apparatus to beprovided with appropriate start-up information so that the appropriatelibraries can be searched, among other things. To render this aspect ofthe operation as automatic as possible, each one of covers 45 providedto the unit is identified by a code plate 85 disposed on an edge of eachcover. The code plate is a relatively simple device which permits eachcover to be readily identified as to test type as well as with otherinformation. One such code plate, and a system of reading the plateusable with the apparatus of FIG. 15, is shown in FIGS. 17-19. In FIG.17 the edge of a cover 29 is provided with a code plate indicatedgenerally at 85 including a plurality of reflective rectangles 86, eachsuch rectangle being adhered to a release paper backing 87 mounted oncover 29. The rectangles can be formed by scribing line 88 at regularintervals in a continuous reflective strip. While only four rectanglesare shown in FIG. 17, it will be apparent that a much larger number ofrectangles can be used, depending upon the number of bits required for aspecific identification purpose. The entire plate can be removed andreplaced for reuse of the cover if desired.

The coding operation takes place by simply removing a selected number ofthe reflective rectangles in specific positions, thereby forming adigital word in which reflective and nonreflective rectangles areestablished in a specific sequence. The rectangles can be arrayed inthis manner in accordance with a preestablished code identifying theoperation to be accomplished with that cover. One partly peeled label isshown at 89. If that label were to be removed completely leaving theother three reflective rectangles in place, and if a system isestablished in which a reflective rectangle represents a binary 1 andthe absence thereof indicates a binary zero, the word formed by theremoval of rectangle 89 would be 1101.

It is contemplated that the code plate would be positioned near thefront end of a cover 29 as shown at 85 in FIG. 13. An optical readerhousing 90 can then be disposed at one end of a guide rail through whichthe cover will pass so that the cover code can be read and theinformation contained therein employed before the Geiger tubes begin togather data from the regions on the cover. A typical optical apparatuswhich can be employed to accomplish this function is shown in FIGS. 18and 19 wherein a housing 90 is mounted on a guide rail 91 and contains asource of light 92, the source of energization for which is conventionaland not shown. A photoelectrical cell 93 is also disposed in housing 90,the source and cell being separated by an opaque barrier 94. An opening95 permits light to emerge from the housing containing source 92 and anopening 96 permits light reflected from code plate 85 to enter thehousing and impinge upon cell 93, producing an electrical variationwhich will be recognized by appropriate photo cell circuitry as theexistence of a digital bit. A sequence of such of bits can be providedto control apparatus 58 by conductors 97 as the cover moves past thereader to initiate the necessary control functions.

Specifically, assume that code 1101 identifies a cover having regions ofcollected CO₂ from an unknown organism which has been incubated with aplurality of known substrates 1 through 36 in table. Receipt of code1101 by control unti 58 will (1) cause that control unit to drive theconveyor past detectors 55 in a stepwise fashion, pausing with eachdetector over its region for a predetermined interval suitable for thattest; (2) indicate to control 82 that the data file set for all countranges resulting from tests with substrates 1 through 36 should be madeavailable for comparison with count data to be provided to store 78; and(3) activate a subroutine to receive each count in multiplexed sequencefrom sequencer 75, compare it with each item in the relevant set in thedata file, and print out the count and match identification whenever amatch occurs, i.e., whenever the count read from a particle detectorfalls within one of the count ranges in the data file set.

This is a relatively simple sequence of events which will yield basicdata on which a first sort can be made. In most cases, this data willindicate a number of organisms which the unknown cannot, or probablywould not, be, and may also indicate most suitable substrates to use ina second set of tests in a second tray. The second test would followsubstantially the same steps as the first except that the codeestablished on plate 85 will indicate to the control unit that adifferent set of substrates is being employed with the unknownmicroorganaism and that a different data file set is to be used forcomparison. This sequence of steps can be repeated, as necessary until asufficiently accurate match with a sufficiently large number ofsubstrates identifies the organism to a required degree of certainty orspecificity.

A more sophisticated, more accurate and faster, but somewhat morecomplex, approach involves the use of the matching and computingcapacity of the computer central processing unit (CPU). In thistechnique, a second data file, which can be a read only memory, isprovided with permutations of matches which identify organisms tovarious degrees of completeness in accordance with known statisticalprocedures. The apparatus can then be queried regarding the identity ofan organism tested on, for example, a set of six trays with anassortment of substrates, to a probability expressed in sigma functions.The resulting printout will then be programmed to yield each suchorganism identity, if more than one exist, which satisfy the requestedaccuracy.

While certain advantageous embodiments of the invention have been chosento illustrate the invention, it will be understood by those skilled inthe art that various changes and modifications can be made thereinwithout departing from the scope of the invention as defined in theappended claims.

I claim:
 1. A process for the rapid identification of a microorganismwhich comprises inoculating a number of different ¹⁴ C labeledsubstrates with an unknown microorganism, at least some of said ¹⁴ Clabeled substrates being capable of being metabolized to ¹⁴ CO₂ bycertain specific microorganisms, incubating said substrates for a timesufficient to cause metabolic breakdown of at least some of saidsubstrates resulting in the production of ¹⁴ CO₂, analyzing forradioactivity in the CO₂ which is evolved from those substrates whichare metabolized to determine which substrates are metabolized and towhat extent by said unknown microorganisms, thereby obtaining asubstrate radiorespirometric profile for said unknown microorganism,comparing said substrate radiorespirometric profile to standardradiorespirometric profiles for known microorganisms obtained by thesame process by which the substrate radiorespirometric profile for saidunknown microorganism was obtained and determining to which standardradiorespirometric profile the radiorespirometric profile for saidunknown microorganism corresponds.
 2. The process as defined in claim 1wherein at least some of said standard substrate radiorespirometricprofiles are obtained by inoculating a number of different ¹⁴ C labeledsubstrates with a known microorganism, at least some of said substratesbeing capable of being metabolized to ¹⁴ CO₂ by said known microorganismincubating all of said substrates for a time sufficient to metabolize atleast some of said substrates resulting in the production of ¹⁴ CO₂,analyzing for evolved radioactive ¹⁴ CO₂ from each of said substrates todetermine which substrates have been metabolized by said knownmicroorganisms and to what extent, thereby establishing a standardradiorespirometric profile for said known microorganism.
 3. The processas defined in claim 1 wherein at least some of said standard substrateradiorespirometric profiles are obtained by inoculating a number ofdifferent ¹⁴ C labeled substrates with an unknown microorganism, atleast some of said substrates being capable of being metabolized to ¹⁴CO₂ by said unknown microorganism, incubating all of said substrates fora time sufficient to metabolize at least some of said substratesresulting in the production of ¹⁴ CO₂, analyzing for evolved radioactive¹⁴ CO₂ from each of said substrates to determine which substrates havebeen metabolized by said unknown microorganism and to what extent,thereby establishing a standard radiorespirometric profile for saidunknown microorganism and thereafter identifying said unknownmicroorganism by biochemical methods.
 4. The process as defined in claim1 wherein said microorganism is identified by genus.
 5. The process asdefined in claim 1 wherein said microorganism is identified as aparticular species within a genus.
 6. The process as defined in claim 1wherein said substrates do not require the presence of inducible enzymesfor metabolism to take place.
 7. The process as defined in claim 1wherein each of said substrates contains at least 4 nanocuries ofradioactivity.
 8. The process as defined in claim 1 wherein each of saidinoculated substrates is incubated for from 5 minutes to 4 hours.
 9. Theprocess as defined in claim 1 wherein any ¹⁴ CO₂ which is evolved fromeach substrate during incubation is collected by means of a getter card.10. The process as defined in claim 9 wherein any ¹⁴ CO₂ evolved fromeach substrate is measured by assaying for radioactivity by means of aninstrument which contains Geiger tubes.
 11. The process as defined inclaim 1 wherein said unknown microorganism is an isolation specimenwhich contains inhibitors for the growth of all microorganisms exceptcertain species.
 12. The process as defined in claim 1 wherein the totalvolume of inoculum plus substrate is between 0.05 to 0.2 ml.